Nejvíce citovaný článek - PubMed ID 36637991
Combined in vitro and cell-based selection display method producing specific binders against IL-9 receptor in high yields
Here, we present a previously undescribed approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated that the nucleotides immediately following the start codon can significantly influence protein expression. However, the impact of these sequences is construct-specific and is not universally applicable to all proteins. Most of the previous research has been limited to selecting from a few rationally designed sequences. In contrast, we used a directed evolution-based methodology, screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increased expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.
- Klíčová slova
- DNA libraries, N‐terminal sequences, directed evolution, fluorescence‐activated cell sorting (FACS), protein expression optimization,
- MeSH
- Escherichia coli * genetika metabolismus MeSH
- genová knihovna * MeSH
- klonování DNA MeSH
- průtoková cytometrie * metody MeSH
- rekombinantní proteiny * genetika biosyntéza MeSH
- řízená evoluce molekul MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- rekombinantní proteiny * MeSH
- zelené fluorescenční proteiny MeSH
Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.
